Compositions isolated from bovine tissues and methods for their use

ABSTRACT

Isolated polynucleotides encoding histatin polypeptides expressed in bovine tissues, including mammary gland and paratoid salivary gland tissues, are provided, together with genetic constructs and host cells comprising such isolated polynucleotides. Methods for the use of such polynucleotides and polypeptides are also provided.

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is a continuation-in-part of U.S. PatentApplication No. 09/699,146, filed Oct. 27, 2000, which claims priorityto U.S. Provisional Patent Application No. 60/162,701, filed Oct. 29,1999; and is a continuation-in-part of U.S. patent application Ser. No.09/644,190, filed Aug. 22, 2000, which claims priority to U.S.Provisional Patent Application No. 60/150,330, filed Aug. 23, 1999.

TECHNICAL FIELD OF THE INVENTION

[0003] This invention relates generally to polypeptides expressed inbovine tissues, including mammary gland cells and paratoid salivarygland cells, to polynucleotides encoding such polypeptides, and tomethods for treating a mammal involving administration of a polypeptideor polynucleotide of the present invention. More particularly, thepresent invention relates to polynucleotides that encode or statherinsand/or histatins, together with polypeptides encoded by suchpolynucleotides and methods for the use of such polypeptides andpolynucleotides.

BACKGROUND OF THE INVENTION

[0004] Human histatins are a family of low molecular weight (51-77 aminoacids), neutral to very basic, histidine-rich, proteins that arespecific to salivary secretions. Histatins are believed to function aspart of the non-immune defense system, particularly in the oral cavity.Salivary histatins have been shown to be potent antifungal,antimicrobial and antibacterial agents, and to have promise astherapeutic agents in humans with oral candidosis (Tsai et al. Infectionand Immunity 64:5000-5007, 1996). In particular, histatins have beenshown to kill the pathogenic yeast, Candida albicans (Oppenheim et al.J. Biol. Chem. 263:7472-7477, 1988), with peptides representing definedportions of the amino acid sequences of naturally occurring human andmacaque histatin being shown to have superior anti-candidal activityover the intact native histatin (Zuo et al. Gene 161:87-91, 1995; U.S.Pat. No. 5,486,503).

[0005] Human statherin is a low molecular weight, acidic phosphoproteinthat acts to inhibit precipitation of calcium phosphate salts in theoral cavity, and which is believed to belong to the same gene family ashuman histatins (Sabatini et al. Mol. Biol. Evol. 10:497-511, 1993).

[0006] Fungal and bacterial infections are common and can be lifethreatening in patients with compromised immune systems. Candidalinfections often occur in diabetics, during pregnancy and followingmedication with antibiotics, steroid hormones or oral contraceptives.Oral candidiasis is common in patients infected with HIV, as well as incancer patients following treatment with radiation or chemotherapy.Systemic fungal infections in immuno-compromised patients and inpatients in intensive care are often fatal since there are few effectiveanti-fungal treatments for intravenous administration. Similarly,bacterial infections can lead to severe disease and even death. Althoughseveral anti-fungal and ant-bacterial agents are available, these agentsare not completely effective, with many not being appropriate for oralor systemic administration. Thus there remains a need in the art forpreparations have effective anti-fungal and anti-bacterial properties.

SUMMARY OF THE INVENTION

[0007] The present invention provides isolated histatin-likepolypeptides expressed in bovine tissues and isolated polynucleotidesencoding such polypeptides, together with genetic constructs and hostcells comprising such polynucleotides. Methods for using suchpolypeptides, polynucleotides and genetic constructs are also provided.

[0008] In specific embodiments, isolated polynucleotides are providedthat comprise a polynucleotide sequence selected from the groupconsisting of: (a) sequences recited in SEQ ID NOS: 1-6; (b) complementsof the sequences recited in SEQ ID NOS: 1-6; (c) reverse complements ofthe sequences recited in SEQ ID NOS: 1-6; (d) reverse sequences of thesequences recited in SEQ ID NOS: 1-6; (e) sequences having a 99%probability of being to the same as a sequence of (a)-(d), determined asdescribed below; and (f) sequences having at least 75%, 90% or 95%identity to a sequence of (a)-(d), the percentage identity beingdetermined as described below. Polynucleotides comprising at least aspecified number of contiguous residues (“x-mers”) of any of thesequences identified as SEQ ID NOS: 1-6 are also provided, together withextended sequences, and oligonucleotide probes and primers correspondingto the sequences set out in SEQ ID NOS: 1-6. All of thesepolynucleotides and oligonucleotide probes and primers are collectivelyreferred to herein as “polynucleotides of the present invention”.

[0009] In further embodiments, the present invention provides isolatedpolypeptides comprising an amino acid sequence encoded by apolynucleotide selected from the group consisting of: (a) sequencesprovided in SEQ ID NOS: 1-6; and (b) sequences having at least 75%, 90%or 95% identity to a sequence provided in SEQ ID NOS: 1-6. In specificembodiments, such polypeptides comprise a sequence selected from thegroup consisting of sequences identified as SEQ ID NO: 7-12, andvariants thereof. The present invention further provides isolatedpolypeptides comprising at least a functional portion of an amino acidsequence encoded by a polynucleotide selected from the group consistingof: (a) sequences provided in SEQ ID NOS: 1-6; and (b) sequences having75%, 90% or 95% identity to a sequence of SEQ ID NOS: 1-6.

[0010] In related embodiments, the present invention provides geneticconstructs comprising the inventive polynucleotides, together with hostcells transformed with such constructs, and organisms comprising suchhost cells.

[0011] In a further aspect, the present invention provides compositionscomprising an inventive polypeptide or polynucleotides in combinationwith a physiologically acceptable carrier and/or an immunostimulant,together with methods for the use of such compositions in the treatmentof a disorder in a mammal, such as a cow or a human. Preferably thedisorder is selected from the group consisting of bacterial, microbialand fungal infections. Such infections may be either local or systemic.In certain embodiments, anti-microbial cosmetic compositions comprisingthe inventive polypeptides are provided.

[0012] The isolated polynucleotides of the present invention havefurther utility in genome mapping, in physical mapping, and inpositional cloning of genes. Additionally, the polynucleotide sequencesidentified as SEQ ID NOS: 1-6, and their variants, may be used to designoligonucleotide probes and primers (referred to collectively as“oligonucleotides”). As detailed below, oligonucleotide probes andprimers have sequences that are substantially complementary to thepolynucleotide of interest over a certain portion of the polynucleotide.The inventive oligonucleotide probes may be used to detect the presence,and examine the expression patterns, of genes in any organism havingsufficiently similar DNA and RNA sequences in their cells usingtechniques that are well known in the art, such as slot blot DNAhybridization techniques. The inventive oligonucleotide primers may beused for PCR amplifications. Oligonucleotide probes and primers of thepresent invention may also be used in connection with various microarraytechnologies, including the microarray technology of Affymetrix, Inc.(Santa Clara, Calif.).

[0013] The above-mentioned and additional features of the presentinvention, together with the manner of obtaining them, will be bestunderstood by reference to the following more detailed description. Allreferences disclosed herein are incorporated herein by reference intheir entirety as if each was incorporated individually.

DETAILED DESCRIPTION OF THE INVENTION

[0014] In certain aspects, the present invention providespolynucleotides that were isolated by sequencing of cDNA libraries frombovine tissues, including mammary gland cells and paratoid salivarygland cells, together with isolated polypeptides encoded by suchpolynucleotides. The polynucleotides of the present invention encodepolypeptides that show similarities to both histatins and statherins. Asdiscussed above, histatins have been shown to possess toxic activityagainst a large range of organisms, including oral bacterial and Candidaalbicans. Accordingly, the inventive polypeptides may be employed in thetreatment of fungal and bacterial infections of the oral cavity, vagina,urethra, ear, skin, respiratory tract, mucosa and eye, as well in thetreatment of systemic infections.

[0015] Organisms against which the inventive compositions may beeffectively employed include, but are not limited to: Candida albicans(both blastospore & mycelial forms), Actinomyces actonimycetemcomitans,Actinomyces viscosus, Bacteroides forsythus, Bacteroides fragilis,Bacteroides gracilis, Bacteroides ureolyticus, Campylobacter concisus,Campylobacter rectus, Campylobacter showae, Campylobacter sputorum,Capnocytophaga gingivalis, Capnocytophaga ochracea, Capnocytophagasputigena, Clostridium histolyticum, Eikenella corrodens, Eubacteriumnodatum, Fusobacterium nucleatum, Fusobacterium periodonticum,Peptostreptococcus micros, Porphyromonas endodontalis, Porphyromonasgingivalis, Prevotella intermedia, Prevotella nigrescens,Propionibacterium acnes, Pseudomonas aeruginosa, Selenomonas noxia,Staphylcoccus aureus, Streptococcus constellatus, Streptococcusgordonii, Streptococcus intermedius, Streptococcus mutans, Streptococcusoralis, Streptococcus pneumonia, Streptococcus sanguis, Treponemadenticola, Treponema pectinvorum, Treptonema socranskii, Veillonellaparvula, and Wolinella succinogenes.

[0016] The inventive polypeptides may be usefully employed in thetreatment of systemic fungal and/or bacterial infections, such assystemic Candida infections in individuals who have compromised immunesystems. The polypeptides disclosed herein may also be employed in thetreatment of dental caries, as well as anti-plaque and anti-tartaragents, and may be delivered by incorporation into toothpastes ormouthwashes. In addition, the inventive polypeptides may be used asanti-microbial agents in cosmetic preparations such as deodorants,soaps, shampoos, etc. Compositions disclosed herein may be used in thetreatment of disorders involving infection of the pulmonary region withPseudomonas aeruginosa, including cystic fibrosis. For use in suchmethods, the compositions may be delivered by a spray mechanism.

[0017] The invention polypeptides and polynucleotides may also beemployed in veterinary applications, including, but not limited to,treatment of mastitis.

[0018] Isolated polynucleotides of the present invention include thepolynucleotides identified herein as SEQ ID NOS: 1-6; isolatedpolynucleotides comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NOS: 1-6; isolated polynucleotides comprisingat least a specified number of contiguous residues (x-mers) of any ofthe polynucleotides identified as SEQ ID NOS: 1-6; isolatedpolynucleotides comprising a polynucleotide sequence that iscomplementary to any of the above polynucleotides; isolatedpolynucleotides comprising a polynucleotide sequence that is a reversesequence or a reverse complement of any of the above polynucleotides;antisense sequences corresponding to any of the above polynucleotides;and variants of any of the above polynucleotides, as that term isdescribed in this specification.

[0019] The definition of the terms “complement,” “reverse complement,”and “reverse sequence,” as used herein, is best illustrated by thefollowing example. For the sequence 5′AGGACC 3′, the complement, reversecomplement, and reverse sequence are as follows: complement 3′ TCCTGG 5′reverse complement 3′ GGTCCT 5′ reverse sequence 5′ CCAGGA 3′.

[0020] Preferably, sequences that are complements of a specificallyrecited polynucleotide sequence are complementary over the entire lengthof the specific polynucleotide sequence.

[0021] As used herein, the term “oligonucleotide” refers to a relativelyshort segment of a polynucleotide sequence, generally comprising between6 and 60 nucleotides, and comprehends both probes for use inhybridization assays and primers for use in the amplification of DNA bypolymerase chain reaction.

[0022] As used herein, the term “polynucleotide” means a single- ordouble-stranded polymer of deoxyribonucleotide or ribonucleotide basesand includes DNA and RNA molecules, both sense and anti-sense strands.The term comprehends cDNA, genomic DNA, recombinant DNA, and wholly orpartially synthesized nucleic acid molecules. A polynucleotide mayconsist of an entire gene, or a portion thereof. A gene is a DNAsequence that codes for a functional protein or RNA molecule. Operableanti-sense polynucleotides may comprise a fragment of the correspondingpolynucleotide, and the definition of “polynucleotide” thereforeincludes all operable anti-sense fragments. Anti-sense polynucleotidesand techniques involving anti-sense polynucleotides are well known inthe art and are described, for example, in Robinson-Benion et al.,“Anti-sense techniques,” Methods in Enzymol. 254(23):363-375, 1995; andKawasaki et al., Artific. Organs 20(8):836-848, 1996.

[0023] Identification of genomic DNA and heterologous species DNA can beaccomplished by standard DNA/DNA hybridization techniques, underappropriately stringent conditions, using all or part of apolynucleotide sequence as a probe to screen an appropriate library.Alternatively, PCR techniques using oligonucleotide primers that aredesigned based on known genomic DNA, cDNA and protein sequences can beused to amplify and identify genomic and/or cDNA sequences. Syntheticpolynucleotides corresponding to the identified sequences, and variantsthereof, may be produced by conventional synthesis methods.

[0024] All the polynucleotides and polypeptides provided by the presentinvention are isolated and purified, as those terms are commonly used inthe art. Preferably, the inventive polypeptides and polynucleotides areat least about 80% pure, more preferably at least about 90% pure, andmost preferably at least about 99% pure.

[0025] The polynucleotide sequences identified as SEQ ID NOS: 1-6 werederived from bovine tissue. Certain of the isolated polynucleotides,including those of SEQ ID NO: 1-4 and 6, disclosed herein arefull-length sequences in that they contain open reading frames. However,certain of the polynucleotides of the present invention may be “partial”sequences, in that they do not represent a full-length gene encoding afull-length polypeptide. Such partial sequences may be extended byanalyzing and sequencing various DNA libraries using primers and/orprobes and well known hybridization and/or PCR techniques.Polynucleotide sequences disclosed herein may thus be extended until anopen reading frame encoding a polypeptide, a full-length polynucleotideand/or gene capable of expressing a polypeptide, or another usefulportion of the genome is identified. Such extended sequences, includingfull length polynucleotides and genes, are described as “correspondingto” a sequence identified as one of the sequences of SEQ ID NOS: 1-6, ora variant thereof, or a portion of one of the sequences of SEQ ID NOS:1-6, or a variant thereof, when the extended polynucleotide comprises anidentified sequence or its variant, or an identified contiguous portion(x-mer) of one of the sequences of SEQ ID NOS: 1-6 or a variant thereof.

[0026] The polynucleotides identified as SEQ ID NOS: 1-6 were isolatedfrom bovine cDNA libraries and represent sequences that are expressed inthe tissue from which the cDNA was prepared. The sequence informationmay be used to isolate or synthesize expressible DNA molecules, such asopen reading frames or full-length genes, that can then be used asexpressible or otherwise functional DNA in cows and other organisms.Similarly, RNA sequences, reverse sequences, complementary sequences,antisense sequences and the like, corresponding to the polynucleotidesof the present invention, may be routinely ascertained and obtainedusing the cDNA sequences identified as SEQ ID NOS: 1-6.

[0027] The polynucleotides identified as SEQ ID NOS: 1-6 may containopen reading frames (“ORFs”) or partial open reading frames encodingpolypeptides. Additionally, open reading frames encoding polypeptidesmay be identified in extended or full-length sequences corresponding tothe sequences set out as SEQ ID NOS: 1-6. Open reading frames may beidentified using techniques that are well known in the art. Thesetechniques include, for example, analysis for the location of knownstart and stop codons, most likely reading frame identification based oncodon frequencies, etc. Suitable tools and software for ORF analysis areavailable, for example, on the Internet at the National Institutes ofHealth NCBI website. Additional tools and software for ORF analysis areavailable, and include GeneWise, available from The Sanger Center,Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UnitedKingdom; Diogenes, available from Computational Biology Centers,University of Minnesota, Academic Health Center, UMHG Box 43 MinneapolisMinn. 55455; and GRAIL, available from the Informatics Group, Oak RidgeNational Laboratories, Oak Ridge, Tennessee Tenn. Open reading framesand portions of open reading frames may be identified in thepolynucleotides of the present invention. Once a partial open readingframe is identified, the polynucleotide may be extended in the area ofthe partial open reading frame using techniques that are well known inthe art until the polynucleotide for the full open reading frame isidentified. Thus, polynucleotides and open reading frames encodingpolypeptides may be identified using the polynucleotides of the presentinvention.

[0028] Once open reading frames are identified in the polynucleotides ofthe present invention, the open reading frames may be isolated and/orsynthesized. Expressible genetic constructs comprising the open readingframes and suitable promoters, initiators, terminators, etc., which arewell known in the art, may then be constructed. Such genetic constructsmay be introduced into a host cell to express the polypeptide encoded bythe open reading frame. Suitable host cells may include variousprokaryotic and eukaryotic cells, including mammalian cells. In vitroexpression of polypeptides is also possible, as well known in the art.

[0029] Polypeptides encoded by the polynucleotides of the presentinvention may be expressed and used in various assays to determine theirbiological activity. Such polypeptides may be used to raise antibodies,to isolate corresponding interacting proteins or other compounds, and toquantitatively determine levels of interacting proteins or othercompounds.

[0030] In another aspect, the present invention provides isolatedpolypeptides encoded, or partially encoded, by the abovepolynucleotides. As used herein, the term “polypeptide” encompassesamino acid chains of any length, including full-length proteins, whereinthe amino acid residues are linked by covalent peptide bonds. The term“polypeptide encoded by a polynucleotide” as used herein, includespolypeptides encoded by a polynucleotide that comprises an isolatedpolynucleotide sequence or variant provided herein. Polypeptides of thepresent invention may be naturally purified products, or may be producedpartially or wholly using recombinant techniques. Such polypeptides maybe glycosylated with bacterial, fungal, mammalian or other eukaryoticcarbohydrates or may be non-glycosylated. In specific embodiments, theinventive polypeptides comprise an amino acid sequence encoded by apolynucleotide selected from the group consisting of sequences providedin SEQ ID NOS: 1-6, including the amino acid sequences identified as SEQID NO: 7-12, as well as variants of such sequences.

[0031] Polypeptides of the present invention may be producedrecombinantly by inserting a polynucleotide sequence that encodes thepolypeptide into a genetic construct and expressing the polypeptide inan appropriate host. Any of a variety of genetic constructs known tothose of ordinary skill in the art may be employed. Expression may beachieved in any appropriate host cell that has been transformed ortransfected with a genetic construct containing a polynucleotide thatencodes a recombinant polypeptide. Suitable host cells includeprokaryotes, yeast, and higher eukaryotic cells. Preferably, the hostcells employed are Escherichia coli, insect, yeast, or a mammalian cellline such as COS or CHO. The polynucleotide sequences expressed in thismanner may encode naturally occurring polypeptides, portions ofnaturally occurring polypeptides, or other variants thereof.

[0032] In a related aspect, polypeptides are provided that comprise atleast a functional portion of a polypeptide having an amino acidsequence encoded by a polynucleotide of the present invention. As usedherein, the “functional portion” of a polypeptide is that portion whichcontains the active site essential for affecting the function of thepolypeptide, for example, the portion of the molecule that is capable ofbinding one or more reactants. The active site may be made up ofseparate portions present on one or more polypeptide chains and willgenerally exhibit high binding affinity. Based on similarity to knownhistatin polypeptides, including, for example, those disclosed inInternational Patent Publication WO 96/40768, the sequences of SEQ IDNO: 7, 8 and 10 were identified as containing the putitave functionalmotifs, or domains, provided in SEQ ID NO: 13 and 14. Polypeptidescontaining such functional motifs possess anti-microbial activity.

[0033] Functional portions of a polypeptide may also be identified byfirst preparing fragments of the polypeptide by either chemical orenzymatic digestion of the polypeptide, or by mutation analysis of thepolynucleotide that encodes the polypeptide and subsequent expression ofthe resulting mutant polypeptides. The polypeptide fragments or mutantpolypeptides are then tested to determine which portions retainbiological activity, using, for example, the representative assaysprovided below.

[0034] Portions and other variants of the inventive polypeptides mayalso be generated by synthetic or recombinant means. Syntheticpolypeptides having fewer than about 100 amino acids, and generallyfewer than about 50 amino acids, may be generated using techniques wellknown to those of ordinary skill in the art. For example, suchpolypeptides may be synthesized using any of the commercially availablesolid-phase techniques, such as the Merrifield solid-phase synthesismethod, where amino acids are sequentially added to a growing amino acidchain. See Merrifield, J. Am. Chem. Soc. 85:2149-2154, 1963. Equipmentfor automated synthesis of polypeptides is commercially available fromsuppliers such as Perkin Elmer/Applied BioSystems, Inc. (Foster City,Calif.), and may be operated according to the manufacturer'sinstructions. Variants of a native polypeptide may be prepared usingstandard mutagenesis techniques, such as oligonucleotide-directed,site-specific mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA82:488-492, 1985). Sections of polynucleotide sequence may also beremoved using standard techniques to permit preparation of truncatedpolypeptides.

[0035] As used herein, the term “variant” comprehends nucleotide oramino acid sequences different from the specifically identifiedsequences, wherein one or more nucleotides or amino acid residues isdeleted, substituted, or added. Variants may be naturally occurringallelic variants, or non-naturally occurring variants. Variant sequences(polynucleotide or polypeptide) preferably exhibit at least 50%, morepreferably at least 75%, more preferably yet at least 90%, and mostpreferably at least 95% identity to a sequence of the present invention.The percentage identity is determined by aligning the two sequences tobe compared as described below, determining the number of identicalresidues in the aligned portion, dividing that number by the totalnumber of residues in the inventive (queried) sequence, and multiplyingthe result by 100. By way of example only, assume a queriedpolynucleotide having 220 nucleic acids has a hit to a polynucleotidesequence in the EMBL database having 520 nucleic acids over a stretch of23 nucleotides in the alignment produced by the BLASTN algorithm usingthe default parameters as described below. The 23 nucleotide hitincludes 21 identical nucleotides, one gap and one different nucleotide.The percentage identity of the queried polynucleotide to the hit in theEMBL database is thus 21/220 times 100, or 9.5%. The percentage identityof polypeptide sequences may be determined in a similar fashion.

[0036] Polynucleotide and polypeptide sequences may be aligned, andpercentages of identical residues in a specified region may bedetermined against another polynucleotide or polypeptide sequence, usingcomputer algorithms that are publicly available. Two exemplaryalgorithms for aligning and identifying the similarity of polynucleotidesequences are the BLASTN and FASTA algorithms. Polynucleotides may alsobe analyzed using the BLASTX algorithm, which compares the six-frameconceptual translation products of a nucleotide query sequence (bothstrands) against a protein sequence database. The percentage identity ofpolypeptide sequences may be examined using the BLASTP algorithm. TheBLASTN, BLASTP and BLASTX algorithms are available on the NCBI anonymousFTP server under /blast/executables/ and are available from the NationalCenter for Biotechnology Information (NCBI), National Library ofMedicine, Building 38A, Room 8N805, Bethesda, Md 20894, USA. The BLASTNalgorithm Version 2.0.11 [Jan-20-2000], set to the parameters describedbelow, is preferred for use in the determination of polynucleotidevariants according to the present invention. The BLASTP algorithm, setto the parameters described below, is preferred for use in thedetermination of polypeptide variants according to the presentinvention. The use of the BLAST family of algorithms, including BLASTN,BLASTP and BLASTX, is described in the publication of Altschul, et al.,Nucleic Acids Res. 25: 3389-3402, 1997.

[0037] The FASTA and FASTX algorithms are available on the Internet, andfrom the University of Virginia by contacting the Vice Provost forResearch, University of Virginia, P.O. Box 9025, Charlottesville, Va.22906-9025, USA. The FASTA algorithm, set to the default parametersdescribed in the documentation and distributed with the algorithm, maybe used in the determination of polynucleotide variants. The readmefiles for FASTA and FASTX Version 1.0 x that are distributed with thealgorithms describe the use of the algorithms and describe the defaultparameters. The use of the FASTA and FASTX algorithms is described inPearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444-2448, 1988; andPearson, Methods in Enzymol. 183:63-98, 1990.

[0038] The following running parameters are preferred for determinationof alignments and similarities using BLASTN that contribute to the Evalues and percentage identity for polynucleotides: Unix running commandwith the following default parameters: blastall -p blastn -d embldb -e10 -G 0 -E 0 -r 1 -v 30 -b 30 -i queryseq -o results; and parametersare: -p Program Name [String]; -d Database [String]; -e Expectationvalue (E) [Real]; -G Cost to open a gap (zero invokes default behavior)[Integer]; -E Cost to extend a gap (zero invokes default behavior)[Integer]; -r Reward for a nucleotide match (blastn only) [Integer]; -vNumber of one-line descriptions (V) [Integer]; -b Number of alignmentsto show (B) [Integer]; -i Query File [File In]; -o BLAST report OutputFile [File Out] Optional.

[0039] The following running parameters are preferred for determinationof alignments and similarities using BLASTP that contribute to the Evalues and percentage identity of polypeptide sequences: blastall -pblastp -d swissprotdb e 10 -G 0 -E 0 -v 30 -b 30 -i queryseq -o results;the parameters are: -p Program Name [String]; -d Database [String]; -eExpectation value (E) [Real]; -G Cost to open a gap (zero invokesdefault behavior) [Integer]; -E Cost to extend a gap (zero invokesdefault behavior) [Integer]; -v Number of one-line descriptions (v)[Integer]; -b Number of alignments to show (b) [Integer]; -I Query File[File In]; -o BLAST report Output File [File Out] Optional.

[0040] The “hits” to one or more database sequences by a queriedsequence produced by BLASTN, BLASTP, FASTA, or a similar algorithm,align and identify similar portions of sequences. The hits are arrangedin order of the degree of similarity and the length of sequence overlap.Hits to a database sequence generally represent an overlap over only afraction of the sequence length of the queried sequence. The BLASTN,FASTA and BLASTP algorithms also produce “Expect” values forpolynucleotide and polypeptide alignments. The Expect value (E)indicates the number of hits one can “expect” to see over a certainnumber of contiguous sequences by chance when searching a database of acertain size. The Expect value is used as a significance threshold fordetermining whether the hit to a database indicates true similarity. Forexample, an E value of 0.1 assigned to a polynucleotide hit isinterpreted as meaning that in a database of the size of the EMBLdatabase, one might expect to see 0.1 matches over the aligned portionof the sequence with a similar score simply by chance. By thiscriterion, the aligned and matched portions of the sequences then have aprobability of 90% of being related. For sequences having an E value of0.01 or less over aligned and matched portions, the probability offinding a match by chance in the EMBL database is 1% or less using theBLASTN algorithm. E values for polypeptide sequences may be determinedin a similar fashion using various polypeptide databases, such as theSwissProt database.

[0041] According to one embodiment, “variant” polynucleotides andpolypeptides, with reference to each of the polynucleotides andpolypeptides of the present invention, preferably comprise sequenceshaving the same number or fewer nucleic or amino acids than each of thepolynucleotides or polypeptides of the present invention and producingan E value of 0.01 or less when compared to the polynucleotide orpolypeptide of the present invention. That is, a variant polynucleotideor polypeptide is any sequence that has at least a 99% probability ofbeing the same as the polynucleotide or polypeptide of the presentinvention, measured as having an E value of 0.01 or less using theBLASTN, FASTA or BLASTP algorithms set at the default parameters.According to a preferred embodiment, a variant polynucleotide is asequence having the same number or fewer nucleic acids than apolynucleotide of the present invention that has at least a 99%probability of being the same as the polynucleotide of the presentinvention, measured as having an E value of 0.01 or less using theBLASTN algorithm set at the default parameters. Similarly, according toa preferred embodiment, a variant polypeptide is a sequence having thesame number or fewer amino acids than a polypeptide of the presentinvention that has at least a 99% probability of being the same as thepolypeptide of the present invention, measured as having an E value of0.01 or less using the BLASTP algorithm set at the default parameters.

[0042] In addition to having a specified percentage identity to aninventive polynucleotide or polypeptide sequence, variantpolynucleotides and polypeptides preferably have additional structureand/or functional features in common with the inventive polynucleotideor polypeptide. Polypeptides having a specified degree of identity to apolypeptide of the present invention share a high degree of similarityin their primary structure and have substantially similar functionalproperties. In addition to sharing a high degree of similarity in theirprimary structure to polynucleotides of the present invention,polynucleotides having a specified degree of identity to, or capable ofhybridizing to, an inventive polynucleotide preferably have at least oneof the following features: (i) they contain an open reading frame orpartial open reading frame encoding a polypeptide having substantiallythe same functional properties as the polypeptide encoded by theinventive polynucleotide; or (ii) they contain identifiable domains incommon.

[0043] In certain embodiments, variant polynucleotides hybridize to apolynucleotide of the present invention under stringent conditions. Asused herein, “stringent conditions” refers to prewashing in a solutionof 633 SSC, 0.2% SDS; hybridizing at 65° C., 6×SSC, 0.2% SDS overnight;followed by two washes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C.and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65° C.

[0044] The present invention also encompasses polynucleotides thatdiffer from the disclosed sequences but that, as a consequence of thediscrepancy of the genetic code, encode a polypeptide having similarenzymatic activity as a polypeptide encoded by a polynucleotide of thepresent invention. Thus, polynucleotides comprising sequences thatdiffer from the polynucleotide sequences recited in SEQ ID NOS: 1-6 (orcomplements, reverse sequences, or reverse complements of thosesequences) as a result of conservative substitutions are encompassedwithin the present invention. Additionally, polynucleotides comprisingsequences that differ from the inventive polynucleotide sequences orcomplements, reverse complements, or reverse sequences as a result ofdeletions and/or insertions totaling less than 10% of the total sequencelength are also contemplated by and encompassed within the presentinvention. Similarly, polypeptides comprising sequences that differ fromthe inventive polypeptide sequences as a result of amino acidsubstitutions, insertions, and/or deletions totaling less than 10% ofthe total sequence length are contemplated by and encompassed within thepresent invention, provided the variant polypeptide has similar activityto the inventive polypeptide.

[0045] The polynucleotides of the present invention may be isolated frombovine cDNA libraries as described below, or may be synthesized usingtechniques that are well known in the art. The polynucleotides may besynthesized, for example, using automated oligonucleotide synthesizers(e.g., Beckman Oligo 1000M DNA Synthesizer) to obtain polynucleotidesegments of up to 50 or more nucleic acids. A plurality of suchpolynucleotide segments may then be ligated using standard DNAmanipulation techniques that are well known in the art of molecularbiology. One conventional and exemplary polynucleotide synthesistechnique involves synthesis of a single stranded polynucleotide segmenthaving, for example, 80 nucleic acids, and hybridizing that segment to asynthesized complementary 85 nucleic acid segment to produce a 5nucleotide overhang. The next segment may then be synthesized in asimilar fashion, with a 5 nucleotide overhang on the opposite strand.The “sticky” ends ensure proper ligation when the two portions arehybridized. In this way, a complete polynucleotide of the presentinvention may be synthesized entirely in vitro.

[0046] As noted above, certain of the polynucleotides identified as SEQID NOS: 1-6 may be referred to as “partial” sequences, in that they maynot represent the full coding portion of a gene encoding a naturallyoccurring polypeptide. Partial polynucleotide sequences disclosed hereinmay be employed to obtain the corresponding full-length genes forvarious species and organisms by, for example, screening DNA expressionlibraries using hybridization probes based on the polynucleotides of thepresent invention, or using PCR amplification with primers based uponthe polynucleotides of the present invention. In this way one can, usingmethods well known in the art, extend a polynucleotide of the presentinvention upstream and downstream of the corresponding mRNA, as well asidentify the corresponding genomic DNA, including the promoter andenhancer regions, of the complete gene. The present invention thuscomprehends isolated polynucleotides comprising a sequence identified inSEQ ID NOS: 1-6, or a variant of one of the specified sequences, thatencode a functional polypeptide, including full-length genes. Suchextended polynucleotides may have a length of from about 50 to about4,000 nucleic acids or base pairs, and preferably have a length of lessthan about 4,000 nucleic acids or base pairs, more preferably yet alength of less than about 3,000 nucleic acids or base pairs, morepreferably yet a length of less than about 2,000 nucleic acids or basepairs. Under some circumstances, extended polynucleotides of the presentinvention may have a length of less than about 1,800 nucleic acids orbase pairs, preferably less than about 1,600 nucleic acids or basepairs, more preferably less than about 1,400 nucleic acids or basepairs, more preferably yet less than about 1,200 nucleic acids or basepairs, and most preferably less than about 1,000 nucleic acids or basepairs.

[0047] As used herein, the term “x-mer,” with reference to a specificvalue of “x,” refers to a polynucleotide or polypeptide, respectively,comprising at least a specified number (“x”) of contiguous residues of:any of the polynucleotides provided in SEQ ID NOS: 1-6. The value of xmay be from about 20 to about 600, depending upon the specific sequence.

[0048] Polynucleotides of the present invention comprehendpolynucleotides comprising at least a specified number of contiguousresidues (x-mers) of any of the polynucleotides identified as SEQ IDNOS: 1-6, or their variants. Polypeptides of the present inventioncomprehend polypeptides comprising at least a specified number ofcontiguous residues (x-mers) of any of the polypeptides corresponding tothe polynucleotides of SEQ ID NOS: 1-6. According to preferredembodiments, the value of x is at least 20, more preferably at least 40,more preferably yet at least 60, and most preferably at least 80. Thus,polynucleotides of the present invention include polynucleotidescomprising a 20-mer, a 40-mer, a 60-mer, an 80-mer, a 100-mer, a120-mer, a 150-mer, a 180-mer, a 200-mer, a 220-mer, a 250-mer, a300-mer, 400-mer, 500-mer or 600-mer of a polynucleotide provided in SEQID NOS: 1-6, or a variant of one of the polynucleotides provided in SEQID NOS: 1-6. Similarly, polypeptides of the present invention includepolypeptides comprising a 20-mer, a 40-mer, a 60-mer, an 80-mer, a100-mer, a 120-mer, a 150-mer, a 180-mer, a 200-mer, a 220-mer, a250-mer, a 300-mer, 400-mer, 500-mer or 600-mer of a polypeptide encodedby a polynucleotide provided in SEQ ID NOS: 1-6, or a variant of one ofthe polynucleotides provided in SEQ ID NOS: 1-6.

[0049] The inventive polynucleotides may be isolated by high throughputsequencing of cDNA libraries prepared from bovine tissue as describedbelow in Example 1. Alternatively, oligonucleotide probes and/or primersbased on the sequences provided in SEQ ID NOS: 1-6, can be synthesizedand used to identify positive clones in either cDNA or genomic DNAlibraries from bovine mammary gland cells by means of hybridization orpolymerase chain reaction (PCR) techniques. Probes can be shorter thanthe sequences provided herein but should be at least about 10,preferably at least about 15 and most preferably at least about 20nucleotides in length. Hybridization and PCR techniques suitable for usewith such oligonucleotide probes are well known in the art (see, forexample, Mullis et al., Cold Spring Harbor Symp. Quant. Biol., 51:263,1987; Erlich, ed., PCR technology, Stockton Press: NY, 1989; andSambrook et al., in Molecular cloning: a laboratory manual, 2nd ed.,CSHL Press: Cold Spring Harbor, N.Y., 1989). Positive clones may beanalyzed by restriction enzyme digestion, DNA sequencing or the like.

[0050] In addition, polynucleotide sequences of the present inventionmay be generated by synthetic means using techniques well known in theart. Equipment for automated synthesis of oligonucleotides iscommercially available from suppliers such as Perkin Elmer/AppliedBiosystems Division (Foster City, Calif.) and may be operated accordingto the manufacturer's instructions.

[0051] Oligonucleotide probes and primers complementary to and/orcorresponding to SEQ ID NOS: 1-6, and variants of those sequences, arealso comprehended by the present invention. Such oligonucleotide probesand primers are substantially complementary to the polynucleotide ofinterest. An oligonucleotide probe or primer is described as“corresponding to” a polynucleotide of the present invention, includingone of the sequences set out as SEQ ID NOS: 1-6 or a variant thereof, ifthe oligonucleotide probe or primer, or its complement, is containedwithin one of the sequences set out as SEQ ID NOS: 1-6 or a variant ofone of the specified sequences.

[0052] Two single stranded sequences are said to be substantiallycomplementary when the nucleotides of one strand, optimally aligned andcompared, with the appropriate nucleotide insertions and/or deletions,pair with at least 80%, preferably at least 90% to 95%, and morepreferably at least 98% to 100%, of the nucleotides of the other strand.Alternatively, substantial complementarity exists when a first DNAstrand will selectively hybridize to a second DNA strand under stringenthybridization conditions. Stringent hybridization conditions fordetermining complementarity include salt conditions of less than about 1M, more usually less than about 500 mM, and preferably less than about200 mM. Hybridization temperatures can be as low as 5° C., but aregenerally greater than about 22° C., more preferably greater than about30° C., and most preferably greater than about 37° C. Longer DNAfragments may require higher hybridization temperatures for specifichybridization. Since the stringency of hybridization may be affected byother factors such as probe composition, presence of organic solvents,and extent of base mismatching, the combination of parameters is moreimportant than the absolute measure of any one alone. DNA-DNAhybridization studies may be performed using either genomic DNA or DNAderived by preparing cDNA from the RNA present in the sample.

[0053] In addition to DNA-DNA hybridization, DNA-RNA or RNA-RNAhybridization assays are also possible. In the first case, the mRNA fromexpressed genes would then be detected instead of genomic DNA or cDNAderived from mRNA of the sample. In the second case, RNA probes could beused. In addition, artificial analogs of DNA hybridizing specifically totarget sequences could also be used.

[0054] In specific embodiments, the inventive oligonucleotide probesand/or primers comprise at least about 6 contiguous residues, morepreferably at least about 10 contiguous residues, and most preferably atleast about 20 contiguous residues complementary to a polynucleotidesequence of the present invention. Probes and primers of the presentinvention may be from about 8 to 100 base pairs in length, or preferablyfrom about 10 to 50 base pairs in length, or more preferably from about15 to 40 base pairs in length. The probes can be easily selected usingprocedures well known in the art, taking into account DNA-DNAhybridization stringencies, annealing and melting temperatures,potential for formation of loops, and other factors which are well knownin the art. Tools and software suitable for designing probes, andespecially suitable for designing PCR primers, are available on theInternet. In addition, a software program suitable for designing probes,and especially for designing PCR primers, is available from PremierBiosoft International, 3786 Corina Way, Palo Alto, Calif. 94303-4504.Preferred techniques for designing PCR primers are also disclosed inDieffenbach and Dyksler, PCR Primer: a laboratory manual, CSHL Press:Cold Spring Harbor, N.Y., 1995.

[0055] A plurality of oligonucleotide probes or primers corresponding toa polynucleotide of the present invention may be provided in a kit form.Such kits generally comprise multiple DNA or oligonucleotide probes,each probe being specific for a polynucleotide sequence. Kits of thepresent invention may comprise one or more probes or primerscorresponding to a polynucleotide of the present invention, including apolynucleotide sequence identified in SEQ ID NOS: 1-6.

[0056] In one embodiment useful for high-throughput assays, theoligonucleotide probe kits of the present invention comprise multipleprobes in an array format, wherein each probe is immobilized in apredefined, spatially addressable location on the surface of a solidsubstrate. Array formats which may be usefully employed in the presentinvention are disclosed, for example, in U.S. Pat. Nos. 5,412,087,5,545,531, and PCT Publication No. WO 95/00530, the disclosures of whichare hereby incorporated by reference.

[0057] Oligonucleotide probes for use in the present invention may beconstructed synthetically prior to immobilization on an array, usingtechniques well known in the art (See, for example, Gait, ed.,Oligonucleotide synthesis a practical approach, IRL Press: Oxford,England, 1984). Automated equipment for the synthesis ofoligonucleotides is available commercially from such companies as PerkinElmer/Applied Biosystems Division (Foster City, Calif.) and may beoperated according to the manufacturer's instructions. Alternatively,the probes may be constructed directly on the surface of the array usingtechniques taught, for example, in PCT Publication No. WO 95/00530.

[0058] The solid substrate and the surface thereof preferably form arigid support and are generally formed from the same material. Examplesof materials from which the solid substrate may be constructed includepolymers, plastics, resins, membranes, polysaccharides, silica orsilica-based materials, carbon, metals and inorganic glasses.Synthetically prepared probes may be immobilized on the surface of thesolid substrate using techniques well known in the art, such as thosedisclosed in U.S. Pat. No. 5,412,087.

[0059] In one such technique, compounds having protected functionalgroups, such as thiols protected with photochemically removableprotecting groups, are attached to the surface of the substrate.Selected regions of the surface are then irradiated with a light source,preferably a laser, to provide reactive thiol groups. This irradiationstep is generally performed using a mask having apertures at predefinedlocations using photolithographic techniques well known in the art ofsemiconductors. The reactive thiol groups are then incubated with theoligonucleotide probe to be immobilized. The precise conditions forincubation, such as temperature, time and pH, depend on the specificprobe and can be easily determined by one of skill in the art. Thesurface of the substrate is washed free of unbound probe and theirradiation step is repeated using a second mask having a differentpattern of apertures. The surface is subsequently incubated with asecond, different, probe. Each oligonucleotide probe is typicallyimmobilized in a discrete area of less than about 1 mm². Preferably eachdiscrete area is less than about 10,000 mm², more preferably less thanabout 100 mm². In this manner, a multitude of oligonucleotide probes maybe immobilized at predefined locations on the array.

[0060] The resulting array may be employed to screen for differences inorganisms or samples or products containing genetic material as follows.Genomic or cDNA libraries are prepared using techniques well known inthe art. The resulting target DNA is then labeled with a suitablemarker, such as a radiolabel, chromophore, fluorophore orchemiluminescent agent, using protocols well known for those skilled inthe art. A solution of the labeled target DNA is contacted with thesurface of the array and incubated for a suitable period of time.

[0061] The surface of the array is then washed free of unbound targetDNA and the probes to which the target DNA hybridized are determined byidentifying those regions of the array to which the markers areattached. When the marker is a radiolabel, such as ³²P, autoradiographyis employed as the detection method. In one embodiment, the marker is afluorophore, such as fluorescein, and the location of bound target DNAis determined by means of fluorescence spectroscopy. Automated equipmentfor use in fluorescence scanning of oligonucleotide probe arrays isavailable from Affymetrix, Inc. (Santa Clara, Calif.) and may beoperated according to the manufacturer's instructions. Such equipmentmay be employed to determine the intensity of fluorescence at eachpredefined location on the array, thereby providing a measure of theamount of target DNA bound at each location. Such an assay would be ableto indicate not only the absence and presence of the marker probe in thetarget, but also the quantitative amount as well.

[0062] In this manner, oligonucleotide probe kits of the presentinvention may be employed to examine the presence/absence (or relativeamounts in case of mixtures) of polynucleotides in different biologicalsamples, or products containing different materials, rapidly and in acost-effective manner.

[0063] Another aspect of the present invention involves collections of aplurality of polynucleotides of the present invention. A collection of aplurality of the polynucleotides of the present invention, particularlythe polynucleotides identified as SEQ ID NOS: 1-6, may be recordedand/or stored on a storage medium and subsequently accessed for purposesof analysis, comparison, etc. Suitable storage media include magneticmedia such as magnetic diskettes, magnetic tapes, CD-ROM storage media,optical storage media, and the like. Suitable storage media and methodsfor recording and storing information, as well as accessing informationsuch as polynucleotide sequences recorded on such media, are well knownin the art. The polynucleotide information stored on the storage mediumis preferably computer-readable and may be used for analysis andcomparison of the polynucleotide information.

[0064] Another aspect of the present invention thus involves storagemedium on which are recorded a collection of the polynucleotides of thepresent invention, particularly a collection of the polynucleotidesidentified as SEQ ID NOS: 1-6. According to one embodiment, the storagemedium includes a collection of at least 20, preferably at least 50,more preferably at least 100, and most preferably at least 200 of thepolynucleotides of the present invention, preferably the polynucleotidesidentified as SEQ ID NOS: 1-6, including variants of thosepolynucleotides.

[0065] In another aspect, the present invention provides geneticconstructs comprising, in the 5′-3′ direction, a gene promoter sequenceand an open reading frame coding for at least a functional portion of apolypeptide encoded by a polynucleotide of the present invention. Incertain embodiments, the genetic constructs of the present inventionalso comprise a gene termination sequence. The open reading frame may beoriented in either a sense or antisense direction. Genetic constructscomprising a non-coding region of a gene coding for a polypeptideencoded by the above polynucleotides or a nucleotide sequencecomplementary to a non-coding region, together with a gene promotersequence, are also provided. A terminator sequence may form part of thisconstruct. Preferably, the gene promoter and termination sequences arefunctional in a host organism. More preferably, the gene promoter andtermination sequences are common to those of the polynucleotide beingintroduced. The genetic construct may further include a marker for theidentification of transformed cells.

[0066] Techniques for operatively linking the components of the geneticconstructs are well known in the art and include the use of syntheticlinkers containing one or more restriction endonuclease sites asdescribed, for example, by Sambrook et al., in Molecular cloning: alaboratory manual, Cold Spring Harbor Laboratories Press: Cold SpringHarbor, N.Y., 1989. The genetic constructs of the present invention maybe linked to a vector having at least one replication system, forexample, E. coli, whereby after each manipulation, the resultingconstruct can be cloned and sequenced and the correctness of themanipulation determined.

[0067] Transgenic cells comprising the genetic constructs of the presentinvention are also provided by the present invention, together withorganisms comprising such transgenic cells, products and progeny of suchorganisms. Techniques for stably incorporating genetic constructs intothe genome of target organisms are well known in the art.

[0068] In one aspect, the present invention provides methods for usingone or more of the inventive polypeptides or polynucleotides to treatdisorders in a subject, preferably a mammal, including, but not limitedto, humans and cows. In this aspect, the polypeptide or polynucleotideis generally present within a composition additionally comprising aphysiologically acceptable carrier.

[0069] Preferred routes of administration for the inventivecompositions, will vary depending on the site and type of disorder to betreated. For example, either systemic or local administration may bepreferred. In general, the compositions may be administered by injection(e.g., intradermal, intramuscular, intravenous, or subcutaneous);intranasally (e.g., by aspiration); orally; or topically. Similarly, thepreferred frequency of administration and dosage will vary fromindividual to individual, and can be readily determined by one of skillin the art using standard techniques. In general, the amount ofpolypeptide present in a dose (or produced in situ by the DNA in a dose)ranges from about 1 pg to about 100 mg per kg of host, typically fromabout 10 pg to about 1 mg per kg of host, and preferably from about 100pg to about 1 μg per kg of host. Suitable dose sizes will vary with thesize of the mammal, but will typically range from about 0.1 ml to about5 ml.

[0070] While any suitable carrier known to those of ordinary skill inthe art may be employed in the compositions of this invention, thepreferred type of carrier will vary depending on the mode ofadministration. For parenteral administration, such as subcutaneousinjection, the carrier preferably comprises water, saline, alcohol, alipid, a wax, or a buffer. For oral administration, any of the abovecarriers or a solid carrier, such as mannitol, lactose, starch,magnesium stearate, sodium saccharine, talcum, cellulose, glucose,sucrose, and magnesium carbonate, may be employed. Biodegradablemicrospheres (e.g., polylactic galactide) may also be employed ascarriers for the pharmaceutical compositions of this invention. Suitablebiodegradable microspheres are disclosed, for example, in U.S. Pat. Nos.4,897,268 and 5,075,109. The compositions of the present invention mayinclude additional components, such as anti-inflammatory agents andknown anti-bacterial agents. When used as anti-microbial cosmeticcompositions, the inventive compositions may contain, for example,binders and thickeners, flavors and/or perfumes as taught in U.S. Pat.No. 5,672,351.

[0071] A composition of the present invention may contain apolynucleotide encoding one or more polypeptides as described above,such that the polypeptide is generated in situ. In such compositions,the polynucleotide may be present within any of a variety of deliverysystems known to those of ordinary skill in the art, including nucleicacid expression systems, and bacterial and viral expression systems.Appropriate nucleic acid expression systems contain the necessarypolynucleotide sequences for expression in a mammal (such as a suitablepromoter and terminator signal). Bacterial delivery systems involve theadministration of a bacterium (such as Bacillus Calmette-Guerin) thatexpresses an immunogenic portion of the polypeptide on its cell surface.In a preferred embodiment, the DNA may be introduced using a viralexpression system (e.g., vaccinia or other poxvirus, retrovirus, oradenovirus), which may involve the use of a non-pathogenic, ordefective, replication competent virus. Techniques for incorporatingpolynucleotides into such expression systems are well known in the art.The DNA may also be “naked,” as described, for example, in Ulmer etal.,Science 259:1745-1749, 1993; and reviewed by Cohen, Science259:1691-1692, 1993. The uptake of naked DNA may be increased by coatingthe DNA onto biodegradable beads, which are efficiently transported intothe cells.

[0072] In certain embodiments, the inventive compositions comprise apolypeptide or polynucleotide disclosed herein in combination with animmunostimulant, such as an adjuvant. Any of a variety ofimmunostimulants may be employed in such compositions tonon-specifically enhance the immune response. Most adjuvants contain asubstance designed to protect the antigen from rapid catabolism, such asaluminum hydroxide or mineral oil, and a non-specific stimulator ofimmune responses, such as lipid A, Bordetella pertussis, orMycobacterium tuberculosis. Suitable adjuvants are commerciallyavailable as, for example, Freund's Incomplete Adjuvant and Freund'sComplete Adjuvant (Difco Laboratories, Detroit, Mich.), and MerckAdjuvant 65 (Merck and Company, Inc., Rahway, N.J.). Other suitableadjuvants include alum, biodegradable microspheres, monophosphoryl lipidA, and Quil A.

[0073] The polypeptides of the present invention may additionally beused in assays to determine biological activity, to raise antibodies, toisolate corresponding ligands or receptors, and in assays toquantitatively determine levels of protein or cognate correspondingligand or receptor, as anti-inflammatory agents.

[0074] The polynucleotides of the present invention may be used forexpression in a transgenic animal, as disclosed in U.S. Pat. No.5,714,345, which teaches the use of transgenic animals capable ofexpressing a desired protein prepared by introducing into an egg orembryo cell of an animal, an expression construct containing thesequence corresponding at least in part to a specific polynucleotide,which encodes the desired protein. In the same manner, the desiredprotein corresponding to a selected polynucleotide sequence of thepresent invention, may be employed in transgenic animals for theproduction of milk containing the desired protein, as disclosed in U.S.Pat. No. 5,849,992.

[0075] In addition, the regulatory sequences contained in the inventivepolynucleotide sequences, or regulatory sequences isolated using theinventive sequences for genome screening and sequencing, as well knownin the art, may be used in transgenic animals to direct the expressionof a desired gene product according to the nature of the regulatorypolynucleotide sequence, in a way similar to that taught in U.S. Pat.No. 5,850,000.

EXAMPLE 1 Isolation of cDNA Sequences from Bovine cDNA Libraries

[0076] Polynucleotides were isolated from bovine mammary gland andparatoid salivary gland cDNA libraries, from a normalized bovine embryocDNA library and from a normalized bovine library as follows.

[0077] Bovine mammary gland cDNA expression libraries were constructedand screened as follows. mRNA was extracted from lactating bovinemammary tissue (late lactating, non-pregnant Jersey, 2 hourspost-milking) using standard protocols. mRNA was precipitated withethanol and the total RNA preparate was purified using a Poly(A) QuikMRNA Isolation Kit (Stratagene, La Jolla, Calif.). A cDNA expressionlibrary was constructed from the purified mRNA by reverse transcriptasesynthesis followed by insertion of the resulting cDNA clones in LambdaZAP using a ZAP Express cDNA Synthesis Kit (Stratagene), according tothe manufacturer's protocol. cDNA expression libraries were preparedfrom bovine paratoid salivary gland tissue and from bovine embryo tissueessentially as described above. In addition, a normalized cDNAexpression library (referred to as the normalized bovine library) wasprepared from a pool of bovine embryo, immunological, placental andmammary tissue, also essentially as described above.

[0078] The resulting cDNAs were packaged using a Gigapack II PackagingExtract (Stratagene) employing 1 μl of sample DNA from the 5 μl ligationmix. Mass excision of the library was done using XL1-Blue MRF' cells andXLOLR cells (Stratagene) with ExAssist helper phage (Stratagene). Theexcised phagemids were diluted with NZY broth (Gibco BRL, Gaithersburg,Md.) and plated out onto LB-kanamycin agar plates containing5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal) andisopropylthio-beta-galactoside (IPTG).

[0079] Of the colonies plated and picked for DNA preparations, the largemajority contained an insert suitable for sequencing. Positive colonieswere cultured in NZY broth with kanamycin and cDNA was purified by meansof REAL DNA minipreps (Qiagen, Venlo, The Netherlands). Agarose gel at1% was used to screen sequencing templates for chromosomalcontamination. Dye terminator sequences were prepared using a Biomek2000 robot (Beckman Coulter Inc., Fullerton, Calif.) for liquid handlingand DNA amplification using a 9700 PCR machine (Perkin Elmer/AppliedBiosystems, Foster City, Calif.) according to the manufacturer'sprotocol.

[0080] The DNA sequences for positive clones were obtained using aPerkin Elmer/Applied Biosystems Division Prism 377 sequencer. cDNAclones were sequenced from the 5′ end. The sequences of polynucleotidesisolated from mammary gland cDNA libraries are identified as SEQ ID NOS:1 and 6. The cDNA sequence identified as SEQ ID NO: 2 was isolated fromthe normalized bovine library, with the sequence of SEQ ID NO: 3 beingisolated from the normalized bovine embryo library, and the sequences ofSEQ ID NO: 4 and 5 being isolated from the paratoid salivary glandlibrary. Amino acid sequences corresponding to the cDNA sequences of SEQID NO: 1-6 are provided in SEQ ID NO: 7-12, respectively.

[0081] Based on homology to known sequences, the polynucleotidesequences of SEQ ID NO: 1-6 were identified as encoding bovinehistatins. All these sequences have similar N-termini but differentC-termini. In addition, the amino acid sequence encoded by the DNAsequence of SEQ ID NO: 4 is identical to that encoded by SEQ ID NO: 1,but the two DNA sequences differ in their 5′untranslated regions. Aputative functional histatin anti-microbial domain is present in theamino acid sequences of SEQ ID NO: 7, 8 and 10 at residues 31-38(Lys-Lys-His-His-Arg-Lys-Tyr-Phe; (SEQ ID NO: 14). This domain differsfrom that of the corresponding human histatin homologue in that itcontains an extra amino acid. In addition, the sequences of SEQ ID NO:7, 8 and 10 contain a stratherin-like phosphorylation site about tenamino acids upstream towards the N-terminus. This stratherin motif isnot present in the previously identified human and macaque histatinmolecules. Using standard Northern techniques and 14 different bovinetissues, expression of the bovine histatin disclosed herein was observedonly in mammary and parotid salivary tissue.

[0082] BLASTN Polynucleotide Analysis

[0083] The isolated cDNA sequences were compared to sequences in theEMBL DNA database using the computer algorithm BLASTN. Comparisons ofDNA sequences provided in SEQ ID NOS: 1-6 to sequences in the EMBL DNAdatabase (using BLASTN) were made as of December 2001, using Version2.0.11 [Jan. -20, 2000], and the following Unix runing command: blastall-p blastn -d embldb -e 10 -G0-E0-r 1 -v 30 -b 30-i queryseq -o.

[0084] The sequences of SEQ ID NOS: 1-6 were determined to have lessthan 50% identity, determined as described above, to sequences in theEMBL database using the computer algorithm BLASTN, as described above.

[0085] BLASTP Polypeptide Analysis

[0086] The isolated polypeptide sequences were compared to sequences inthe SwissProt protein database using the computer algorithm BLASTP.Comparisons of polypeptide sequences provided in SEQ ID NOS: 7-12 tosequences in the SwissProt protein database (using BLASTP) were made asof December 2001, using Version 2.0.11 [Jan. 20, 2000], and thefollowing Unix running command: blastall -p blastn -d embldb -e 10-G0-E0-v 30 -b 30 -i queryseq -o. The sequences of SEQ ID NOS: 7-12 weredetermined to have less than 50% identity, determined as describedabove, to sequences in the SwissProt protein database.

[0087] BLASTX Polynucleotide Analysis

[0088] The isolated CDNA sequences were compared to sequences in theSwissProt protein database using the computer algorithm BLASTX.Specifically, comparisons of DNA sequences provided in SEQ ID NOS: 1-6to sequences in the SwissProt protein database (using BLASTP) were madeas of December 2001, using Version 2.0.11 [Jan. 20, 2000], and thefollowing Unix running command: blastall -p blast -d embldb -e 10-G0-E0-v 30 -b 30 -i queryseq o. The sequences of SEQ ID NOS: 1-6 weredetermined to have less than 50% identity, determined as describedabove, to sequences in the SwissProt protein using the computeralgorithm BLASTX, as described above.

EXAMPLE 2 Expression of mRNA in Bovine Mammary Tissue

[0089] This example illustrates the determination of mRNA expressionlevels of mammary-gland specific sequences, isolated essentially asdescribed in Example 1, in biological samples.

[0090] RNA was extracted from mammary gland tissue obtained from anon-pregnant heifer, a pregnant cow and a lactating cow, as well as frombovine liver, forebrain and kidney, using TRIzol (Gibco BRL,Gaithersburg, Md.) following the manufacturer's protocol. Sets of thevarious total RNA samples were run on 1.2% agarose/formaldehyde gels, 5μg/lane. Following transfer to nitrocellulose membranes, RNA wascross-linked with ultraviolet light.

[0091] DNA probes were prepared from bacterial clones transformed withcDNA corresponding to SEQ ID NO: 15 by excision of the insert of thecDNA clone using EcoRI and Xhol restriction endonucleases, or by PCRamplification of the insert of the cDNA clone using T7 and T3 primers(Gibco BRL), or by using the entire cDNA clone. Probes were radiolabeledwith α-P³²-dCTP using Rediprime DNA labeling kits (Amersham PharmaciaBiotech, Uppsala, Sweden). SEQ ID NO: 15 is contained within thesequence of SEQ ID NO: 1.

[0092] Blots were hybridized overnight with rotation at 65° C. in abuffer containing 10-20 ml of 500 mM NaH₂PO₄, 1 mM EDTA, 7% SDS, andthen washed for 15 minutes at 65° C., first in 2×SSC/0.1% and then in0.2×SSC/0.1% SDS. The blots were exposed to Kodak XAR X-ray film forappropriate times.

[0093] The insert of the cDNA clone corresponding to the polynucleotidesequence of SEQ ID NO: 15 hybridized strongly with a transcript ofapproximately 0.8 kb in the lactating mammary gland sample. Very weakhybridization to transcripts of a similar size was detected in themammary samples from a non-pregnant heifer and a pregnant non-lactatingcow. No transcripts could be detected in the other tissue samples.

[0094] SEQ ID NOS: 1-15 are set out in the attached Sequence Listing.The codes for nucleotide sequences used in the attached SequenceListing, including the symbol “n,” conform to WIPO Standard ST.25(1998), Appendix 2, Table 1.

[0095] All references cited herein, including patent references andnon-patent publications, are hereby incorporated by reference in theirentireties.

[0096] While in the foregoing specification this invention has beendescribed in relation to certain preferred embodiments, and many detailshave been set forth for purposes of illustration, it will be apparent tothose skilled in the art that the invention is susceptible to additionalembodiments and that certain of the details described herein may bevaried considerably without departing from the basic principles of theinvention.

1 15 1 505 DNA Bovine 1 ttgaagatga catcaggtac cgctgtccag gactccaccaaatatgaaga tctttatctt 60 tgtcttcatt atggctctca tcctagccat gattagagctgattcatctg aagagaaacg 120 tcacaggaaa cggaaaaaac atcatagagg atattttcaacaataccagc catatcaacg 180 atatccacta aattatcctc ctgcgtatcc atttccttaaaatgctgctt agtaactaca 240 ggacatgatt agagagattt ttcacaatga tttttcctactctttctgtt gtgttgaaaa 300 ccatctttca aatgaataaa acaaagaaaa aaaaatcagtcaagtagttg cacaacacat 360 acttggaatc aaatatcaat attttaaaac ataataatgatagtctctga actatgtaat 420 tggtttctac tttcttttct ctgtcactta ccatgcatgcttaataaatt gatctatcaa 480 gcataaaaaa aaaaaaaaaa aaaaa 505 2 585 DNABovine 2 atggattcga ttggaccaga aaagtggtaa aatcttgatc aaagaaatccttaacaatat 60 actaaagaaa gaagcttgtc caagggaaaa ttaaaaagcc agcaattgaagatgacatca 120 ggtaccgctg tccaggactc caccaaatat gaagatcttt atctttatcttcattatggc 180 tctcatccta gccatgatta gagctgattc atctgaagag aaacgtcacaggaaacggaa 240 aaaacatcat agaggatatt ttcaacaata ccagccatat caacgatatccactaaatta 300 tcctcctgcg tatccatttc cttaaaatgc tgcttagtaa ctacaggacatgattagaga 360 gatttttcac aatgattttt cctactcttt ctgttgtgtt gaaaaccatctttcaaatga 420 ataaaacaaa gaaaaaaaaa tcagtcaagt agttgcacaa cacatacttggaatcaaata 480 tcaatatttt aaaacataat aatgacagtc tctgaactat gtaattggtttctactttct 540 tttctctgtc acttaccatg catgcttaat aaattgatct atcaa 585 3869 DNA Bovine 3 gtccaaggga aaattaaaaa gccagcaatt gaagatgaca tcaggtaccgctgtccagga 60 ctccaccaaa tatgaagatc tttatcttta tcttcattat ggctctcatcctagccatga 120 ttagagctga ttcatctgaa gagaaacgtc acaggaaacg gaaaaaacatcatgtatgta 180 ttcctctgat aatgtggtat agtataagaa tattttttac tcagaatatttattctataa 240 gagaatacat atttatcttt gaaatatatc tatacaatga ttagcttatgtgtccattga 300 attatctttt tatgatacac taggtaaaga ccccaaagac ttgtgtgctgttactgttta 360 catagaaacc tataatgcca ccctgataaa gccagttatt ttctaagaaaagttatttct 420 gtttggtaaa taattgcctt cctctgaaga atactgaaat tctaattatactaagtacaa 480 tatacaccta aaggaccata gaggctgcta aagaacatat attatgtggtcagtttccat 540 tttctgattt cttttgggag gtaatgtatg ttaattgacc aagttaaagaagagagacaa 600 tttcatttat ttaacatttt tcttaatgat atgtatcact ggtgtctcatattattttga 660 catgtctaat caggttgata ggtctccaga attcttacta atacagtaagtactgctact 720 gctactgcta agttgcttca gtcatgtcca actctgtgcg accccatagacagcagcgca 780 ccaggctccc ctgcccctgg gattctccag gcaaggatac tggagtgggttgccatttca 840 gataaattca aatagcttca aaaaaaaaa 869 4 604 DNA Bovine 4gaagtatttt cagttctata ataagatctc ataactgatg taattacaaa aacaaatgaa 60ggatttcaag gtatttaaac acagcagttt tctagcaaag aacatctcct gaagcatcag 120aatttcatct ttcatgactg gactccacca aatatgaaga tctttatctt tgtcttcatt 180atggctctca tcctagccat gattagagct gattcatctg aagagaaacg tcacaggaaa 240cggaaaaaac atcatagagg atattttcaa caataccagc catatcaacg atatccacta 300aattatcctc ctgcgtatcc atttccttaa aatgctgctt agtaactaca ggacatgatt 360agagagattt ttcacaatga tttttcctac tctttctgtt gtgttgaaaa ccatctttca 420aatgaataaa acaaagaaaa aaaaatcagt caagtagttg cacaacacat acttggaatc 480aaatatcaat attttaaaac ataataatga tagtctctga actatgtaat tggtttctac 540tttcttttct ctgtcactta ccatgcatgc ttaataaatt gatctatcaa gcataaaaaa 600aaaa 604 5 96 DNA Bovine 5 tttcaacaat accagccata tgaacgatat ccactaaattatcctcctgc gtatccatta 60 tcttaaaatg ctgcttacca actacaggac atgata 96 6525 DNA Bovine 6 tgaagatgac atcaggtacc gctgtccagg actccaccaa atatgaagatctttatcttt 60 gtcttcatta tggctctcat cctagccatg attagagctg attcatctgaagagaaacgt 120 cacaggaaac ggaaaaaaca tcatgttgat aggtctccag aattcttactaatacaagag 180 gatattttca acaataccag ccatatcaac gatatccact aaattatcctcctgcgtatc 240 catttcctta aaatgctgct tagtaactac aggacatgat tagagagatttttcacaatg 300 atttttccta ctctttctgt tgtgttgaaa accatctttc aaatgaataaaacaaagaaa 360 aaaaaatcag tcaagtagtt gcacaacaca tacttggaat caaatatcaatattttaaaa 420 cataataatg atagtctctg aactatgtaa ttggtttcta ctttcttttctctgtcactt 480 accatgcatg cttaataaat tgatctatca agcataaaaa aaaaa 525 758 PRT Bovine 7 Met Lys Ile Phe Ile Phe Val Phe Ile Met Ala Leu Ile LeuAla Met 1 5 10 15 Ile Arg Ala Asp Ser Ser Glu Glu Lys Arg His Arg LysArg Lys Lys 20 25 30 His His Arg Gly Tyr Phe Gln Gln Tyr Gln Pro Tyr GlnArg Tyr Pro 35 40 45 Leu Asn Tyr Pro Pro Ala Tyr Pro Phe Pro 50 55 8 58PRT Bovine 8 Met Lys Ile Phe Ile Phe Ile Phe Ile Met Ala Leu Ile Leu AlaMet 1 5 10 15 Ile Arg Ala Asp Ser Ser Glu Glu Lys Arg His Arg Lys ArgLys Lys 20 25 30 His His Arg Gly Tyr Phe Gln Gln Tyr Gln Pro Tyr Gln ArgTyr Pro 35 40 45 Leu Asn Tyr Pro Pro Ala Tyr Pro Phe Pro 50 55 9 70 PRTBovine 9 Met Lys Ile Phe Ile Phe Ile Phe Ile Met Ala Leu Ile Leu Ala Met1 5 10 15 Ile Arg Ala Asp Ser Ser Glu Glu Lys Arg His Arg Lys Arg LysLys 20 25 30 His His Val Cys Ile Pro Leu Ile Met Trp Tyr Ser Ile Arg IlePhe 35 40 45 Phe Thr Gln Asn Ile Tyr Ser Ile Arg Glu Tyr Ile Phe Ile PheGlu 50 55 60 Ile Tyr Leu Tyr Asn Asp 65 70 10 58 PRT Bovine 10 Met LysIle Phe Ile Phe Val Phe Ile Met Ala Leu Ile Leu Ala Met 1 5 10 15 IleArg Ala Asp Ser Ser Glu Glu Lys Arg His Arg Lys Arg Lys Lys 20 25 30 HisHis Arg Gly Tyr Phe Gln Gln Tyr Gln Pro Tyr Gln Arg Tyr Pro 35 40 45 LeuAsn Tyr Pro Pro Ala Tyr Pro Phe Pro 50 55 11 21 PRT Bovine 11 Phe GlnGln Tyr Gln Pro Tyr Glu Arg Tyr Pro Leu Asn Tyr Pro Pro 1 5 10 15 AlaTyr Pro Leu Ser 20 12 59 PRT Bovine 12 Met Lys Ile Phe Ile Phe Val PheIle Met Ala Leu Ile Leu Ala Met 1 5 10 15 Ile Arg Ala Asp Ser Ser GluGlu Lys Arg His Arg Lys Arg Lys Lys 20 25 30 His His Val Asp Arg Ser ProGlu Phe Leu Leu Ile Gln Glu Asp Ile 35 40 45 Phe Asn Asn Thr Ser His IleAsn Asp Ile His 50 55 13 8 PRT Artificial Sequence Made in a lab fromsynthetic material 13 Xaa Xaa His His Xaa Xaa Tyr Xaa 1 5 14 8 PRTArtificial Sequence Made in a lab from synthetic material 14 Lys Lys HisHis Arg Lys Tyr Phe 1 5 15 267 DNA Bovine 15 ttgaagatga catcaggtaccgctgtccag gactccacca aatatgaaga tctttatctt 60 tgtcttcatt atggctctcatcctagccat gattagagct gattcatctg aagagaaacg 120 tcacaggaaa cggaaaaaacatcatagagg atattttcaa caataccagc catatcaacg 180 atatccacta aattatcctcctgcgtatcc atttccttaa aatgctgctt agtaactaca 240 ggacatgatt agagagatttttcacaa 267

We claim:
 1. An isolated polynucleotide comprising a sequence selectedfrom the group consisting of: (1) sequences recited in SEQ ID NOS: 1-6;(2) complements of the sequences recited in SEQ ID NOS: 1-6; (3) reversecomplements of the sequences recited in SEQ ID NOS: 1-6; (4) reversesequences of the sequences recited in SEQ ID NOS: 1-6; (5) sequenceshaving at least a 99% probability of being the same as a sequencerecited in (1)-(4) above as determined using computer algorithm BLASTN;(6) sequences having at least 75% identity to a nucleotide sequencerecited in (1)-(4) above determined using computer algorithm BLASTN; (7)sequences having at least 90% identity to a nucleotide sequence recitedin (1)-(4) above determined using computer algorithm BLASTN; (8)sequences having at least 95% identity to a nucleotide sequence recitedin (1)-(4) above determined using computer algorithm BLASTN; (9)nucleotide sequences that hybridize to a sequence recited in (1)-(4)above under stringent hybridization conditions; (10) nucleotidesequences that are 200-mers of a sequence recited in (1)-(4) above; (11)nucleotide sequences that are 100-mers of a sequence recited in (1)-(4)above; (12) nucleotide sequences that are 40-mers of a sequence recitedin (1)-(4) above; (13) nucleotide sequences that are 20-mers of asequence recited in (1)-(4) above; and (14) nucleotide sequences thatare degeneratively equivalent to a sequence recited in (1)-(4) above. 2.An oligonucleotide comprising at least 10 contiguous residuescomplementary to 10 contiguous residues of a nucleotide sequence recitedin claim
 1. 3. A genetic construct comprising an isolated polynucleotideof claim
 1. 4. A host cell transformed with a genetic construct of claim3.
 5. An isolated polypeptide encoded by a polynucleotide of claim
 1. 6.An isolated polypeptide comprising an amino acid sequence selected fromthe group consisting of: (a) sequences provided in SEQ ID NOS: 7-14; (b)sequences having at least a 99% probability of being the same as asequence of SEQ ID NOS: 7-14, as determined using the computer algorithmBLASTP; (c) sequences having at least 75% identity to a sequenceprovided in SEQ ID NOS: 7-14, as determined using the computer algorithmBLASTP; (d) sequences having at least 90% identity to a sequenceprovided in SEQ ID NOS: 7-14, as determined using the computer algorithmBLASTP; and (e) sequences having at least 95% identity to a sequenceprovided in SEQ ID NOS: 7-14, as determined using the computer algorithmBLASTP.
 7. An isolated polynucleotide encoding a polypeptide of claim 6.8. An isolated polypeptide comprising at least a functional portion ofan amino acid sequence selected from the group consisting of: (a)sequences provided in SEQ ID NOS: 7-12; (b) sequences having at least a99% probability of being the same as a sequence of SEQ ID NOS: 7-12, asdetermined using the computer algorithm BLASTP; (c) sequences having atleast 75% identity to a sequence provided in SEQ ID NOS: 7-12, asdetermined using the computer algorithm BLASTP; (d) sequences having atleast 90% identity to a sequence provided in SEQ ID NOS: 7-12, asdetermined using the computer algorithm BLASTP; and (e) sequences havingat least 95% identity to a sequence provided in SEQ ID NOS: 7-12, asdetermined using the computer algorithm BLASTP.
 9. A compositioncomprising a polypeptide according to any one of claims 6 and 8, and atleast one component selected from the group consisting of:physiologically acceptable carriers and immunostimulants.
 10. Acomposition according to claim 9, wherein the composition is a cosmeticcomposition.
 11. A composition comprising a polynucleotide according toclaim 1 and at least one component selected from the group consistingof: pharmaceutically acceptable carriers and immunostimulants.
 12. Amethod for treating a disorder in a mammal, comprising administering acomposition according to claim
 9. 13. The method of claim 12, whereinthe disorder is selected from the group consisting of: microbialinfections and fungal infections.
 14. A method for treating a disorderin a mammal comprising administering a composition according to claim11.
 15. The method of claim 14, wherein the disorder is selected fromthe group consisting of: microbial infections and fungal infections. 16.A transgenic organism comprising a host cell according to claim 4.